Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transfer.
Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transfer.
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通过共振能量转移揭示可溶性低聚物和不溶性聚集体内的构象。
DOI：
10.1002/bip.21324
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Finke,JohnM
中科院分区：
文献类型：
--
作者：
Digambaranath,JyothiL;Dang,Loan;Dembinska,Monika;Vasyluk,Andrew;Finke,JohnM
使用荧光标记的20个残基的聚谷氨酸（polyE）肽20个氨基酸长度的聚谷氨酸（E20）研究E20与其他未标记的polyE肽共聚集时发生的结构变化。使用E20的N-末端的氨基苯甲酰胺供体和C-末端的3-硝基酪氨酸受体进行共振能量转移（RET）。PolyE聚集体不被定义为淀粉样蛋白，因为它们是非纤维状的并且不结合刚果红。圆二色性测量表明，聚E聚集涉及从α-螺旋单体到聚集的β-折叠的转变。可溶性低聚物也沿着反应中的聚集体产生，如通过尺寸排阻色谱法测定的。时间分辨和稳态RET测量揭示了四种主要的E20构象：（1）部分折叠构象（24）单体中的供体-受体距离，（2）可溶性低聚物中的扩展构象（>29倍的供体-受体距离），（3）较小的部分折叠构象（22 π供体-受体距离），和（4）聚集体中的主要高度折叠构象（13 π供体-受体距离）。这些发现证明了RET作为确定可溶性寡聚体和蛋白质聚集状态的埃级结构细节的方法的用途。© 2009威利期刊公司. Biopolymers 93：299-317，2010.这篇文章最初作为公认的预印本在线发表。“在线发布”日期对应于预印本。您可以通过向Biopolymers编辑部发送电子邮件（biopolymers@wiley.com）索取预印本的副本
A fluorescently labeled 20‐residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E20) was used to study structural changes which occur in E20as it co‐aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using ano‐aminobenzamide donor at the N‐terminus and 3‐nitrotyrosine acceptor at the C‐terminus of E20. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from α‐helical monomers to aggregated β‐sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time‐resolved and steady‐state RET measurements reveal four dominant E20conformations: (1) a partially collapsed conformation (24 Å donor–acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 Å donor–acceptor distance), (3) a minor partially collapsed conformation (22 Å donor‐acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 Å donor–acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom‐level structural details of soluble oligomer and aggregated states of proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 299–317, 2010.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com